The research of the Hematopoiesis Section is focused on the basic biology of stem cells and the use of stem cells as vehicles for cell and gene therapy. Hematopoietic stem cells (HSC) are a rare population of self- renewing cells that give rise to all cells in the peripheral blood, making them ideal vehicles for gene replacement therapy of inherited hematopoietic diseases. In addition, cells highly enriched for HSC can generate cardiac myocytes when injected into the healthy tissue surrounding a myocardial infarct or when mobilized into the peripheral blood with cytokines (e.g. G-CSF and SCF). Others have shown that enriched populations of HSC can give rise to vascular, skeletal muscle, hepatic and neuronal cells. Project 1 will examine stem cell biology. Project 1: Biology of Hematopoietic Stem Cells Specific Aim 1.1: We hypothesized that the same hematopoietic stem cells that repopulated the bone marrow were responsible for the regeneration of cardiac myocytes after experimental myocardial infraction (MI). To test this hypothesis, we transplanted mice with bone marrow cells marked with a retrovirus vector containing the GFP gene. These mice then underwent a coronary artery ligation and stem and progenitor cells were mobilized into the peripheral blood with G-CSF and SCF. The infarct region was repopulated with new cells that contain the identical retrovirus markers found in peripheral blood cells. These studies are ongoing, but we conclude that the new cardiac myocytes are the progeny of cells that also give rise to peripheral blood cells. Specific Aim 1.2: We hypothesized that the repopulation of the injured regions of the heart was due to engraftment and expansion of new cells as opposed to fusion of a primitive cell with surrounding cardiac cells. To test this hypothesis we have isolated cells from the regenerating infarct and used in situ hybridization to demonstrate that the cells express proteins in primitive cardiac cells and are diploid, arguing against the fusion hypothesis. Future studies will involve mice genetically deficient in HSC to see whether G-CSF/SCF treatment can repopulate an infarct region. Specific Aim 1.3: We hypothesize that specific genes expressed in both HSC and stem cells isolated from skeletal muscle are responsible for maintaining an undifferentiated state. To test this hypothesis, we have isolated the ?side population? stem cells from mouse skeletal muscle cultures and used cDNA subtraction to generate a library of sequences expressed in these cells. The muscle stem cell transcripts will be compared to those we have identified previously in a cDNA subtraction library of HSC. We will select transcripts common to both libraries for analysis in transgenic mice. Gene transfer to HSC has recently been shown to cure Severe Combined Immune Deficiency, demonstrating that HSC gene therapy could be applied to more common diseases. We would like to develop a gene therapy for Sickle Cell Disease. However, current levels of gene transfer to HSC are too low to treat this disease. We have found that one important reason that gene transfer is so low is that the conventional retrovirus receptors on HSC are nearly undetectable. A second problem has been the instability of retrovirus vectors containing globin genes. Project 2 will examine these problems separately. Project 2: Gene therapy for the hemoglobinopathies Specific aim 2.1: We have evidence that the receptors of the RD114 and FeLV-C retrovirus are expressed at high levels on hematopoietic stem cells. We hypothesize that these high levels of receptor will result in a higher frequency of human HSC transduction. We begun to test this hypothesis by simultaneously transducing human HSC with a control GALV pseudotyped vector and either RD114 or FeLV-C pseudotyped vectors for transplantation into fetal sheep. Peripheral blood and bone marrow cells from the the first 5 sheep chimeras showed greater than 10 fold higher levels of transduction with RD114 or FeLV-C pseudotyped vectors compared to GALV pseudotyped vectors. This work is on going. Specific Aim 2.2: We hypothesize that stable retrovirus vectors containing globin genes linked to the promoters of genes expressed in erythroid cells can be generated that will allow expression of globin mRNA at levels adequate to treat Sickle Cell Disease and b-thalassemia. Our evaluation of the relative level of expression of red cell gene promoters using a transgenic mouse assay has shown that the AE-1 promoter linked to a chicken insulator element directs position independent, uniform, high-level, and copy number dependent expression. We have generated stable retroviruses with this construct which will be evaluated in the mouse b-thalassemia model.